CN102079777A - Artificially synthesized antimicrobial peptide, preparation method and application thereof - Google Patents
Artificially synthesized antimicrobial peptide, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an artificially synthesized antimicrobial peptide, which belongs to the field of biological pharmaceutical technology. The amino acid sequence of the artificially synthesized antimicrobial peptide is Cys-Val-Cys-Phe-Gly-Arg-ARG-Cys-Ile-Cys or Cys-Ile-Cys-Arg-Arg-Gly-Phe-Cys-Val-Cys. The invention also discloses a method for artificially synthesizing the antimicrobial peptide and application of the artificially synthesized antimicrobial peptide in development of preparing medicaments for treating Gram-positive bacteria and Gram-negative bacteria or feed additives. Compared with the prior art, the artificially synthesized antimicrobial peptide has the following advantages and effects, such as small molecular weight as compared with the natural ones, stable structure, strong antimicrobial activity, short sequence, less disulfide bond number, low cost and the like.
Description
Technical field
The invention belongs to field of biological pharmacy, especially relate to artificial synthetic antimicrobial peptide and application thereof.
Background technology
Antibacterial peptide (antimicrobial peptides) has indispensable effect as the integral part of innate immune system for the protection living organism.So far, the separated and evaluation of antibacterial peptide above 800 has been arranged.According to their amino acid form and space structure can be divided into (i) alpha-helix positively charged ion linearity antibacterial peptide, (ii) contain the antibacterial peptide of disulfide linkage, (iii) be rich in the antibacterial peptide of certain amino-acid residue and (iv) negatively charged ion antibacterial peptide.The anti-microbial activity of antibacterial peptide have selectivity height, sterilization fast, effect wide spectrum and be difficult to form the characteristics of resistance, make it become the ideal candidates of research and development new antibiotic.
The mussel antibacterial peptide is an important content in the research of Allopelagic sterilizing peptide, and the structure that it is special and the antibacterial of wide spectrum make it have important theoretical research and be worth, and also having exploitation simultaneously becomes the antibiotic potentiality of new bio.Mytilin is the class important member in the mussel antibacterial peptide family, and separation and purification to 5 kind of mytilin molecule in the mussel at present never of the same race is respectively mytilinA, B, and C, D and G1 are to find the maximum family of member in the present mussel antibacterial peptide research.In view of its high abundance in mussel serum and the broad-spectrum antimicrobial function that shows in the in-vitro antibacterial experiment, mytilin is considered to a most important class antibacterial peptide molecule in the mussel defense mechanism.
The antibacterial of antibacterial peptide and its space structure have substantial connection, therefore, are subjected to people's attention at the structural research of antibacterial peptide always, also are the key points of understanding antibacterial peptide structure and function relationship and antibacterial mechanisms thereof in depth.People such as Roch have carried out nuclear magnetic resonance research to the mytilin B from Mediterranean Sea mussel (Mytilus galloprovincialis) recently, the result shows, the structure of mytilin B belongs to the stable α/beta structure of typical disulfide linkage (Cysteine-stabilized-α/β), include one section alpha-helix and two sections beta-pleated sheets in the molecule, stablize entire structure by four pairs of disulfide linkage.This structure type neither belongs to the common α-Luo Xuanjiegou of antibacterial peptide, does not also belong to beta sheet type antibacterial peptide structure, is the more special space structure of a class, and this similar in people such as Yang in early days to the space structure result of study of mussel alexin MGD-1.And before by the research to structure and the function of MGD-1, Romestand etc. think that by two sections beta-pleated sheets and the positively charged amino-acid residue that connects on the ring (loop) of these two sections beta-pleated sheets be the key position that the mussel antibacterial peptide is brought into play anti-microbial activity.
Mytilus crassitesta Lischke (Mytilus coruscus) is that China has one of breed mussel of important economic worth, in the research in early stage, we from Mytilus crassitesta Lischke serum separation and purification to a kind of mytilin, called after mytilin-1 (Genebank:FJ973154), this antibacterial peptide molecular weight is 3885.3Da, contain 34 amino-acid residues, comprising 8 halfcystines and form 4 pairs of disulfide linkage.In the in-vitro antibacterial experiment, mytilin-1 all has significant inhibitory effect to gram-positive microorganism and Gram-negative bacteria.The mytilin B of Mytilus crassitesta Lischke mytilin-1 and Mediterranean Sea mussel has higher sequence similarity (64%).
At present, extracting natural antibacterial peptide in the organism is the main thought of antibacterial peptide product development, but natural antibacterial peptide belongs to protein because of it, has structural instability, easily shortcoming such as inactivation.
Summary of the invention
The object of the invention be at existing antibacterial peptide structural instability, easily in inactivation, the existing organism antibacterial peptide content extremely low, express by the method for dna recombinant expression and to contain that many long and disulfide linkage is more because of the natural antibacterial peptide sequence makes the synthetic too high defective of cost to disulfide linkage, the method for taking synthetic, design a kind of novel antibacterial peptide according to the active region of Mytilus crassitesta Lischke mytilin, have that molecular weight is little, Stability Analysis of Structures, anti-microbial activity is strong, sequence is short, few, the low cost and other advantages of disulfide linkage number.
For achieving the above object, the present invention adopts following technical scheme:
A kind of artificial synthetic antimicrobial peptide, its aminoacid sequence are Cys-Val-Cys-Phe-Gly-Arg-Arg-Cys-Ile-Cys; Perhaps be Cys-Ile-Cys-Arg-Arg-Gly-Phe-Cys-Val-Cys.
Preferred scheme is: comprise two pairs of disulfide linkage in the described antibacterial peptide aminoacid sequence, described antibacterial peptide has β-hairpin structure.
More excellent scheme is: described antibacterial peptide is one or several amino acid whose being substituted in the antibacterial peptide aminoacid sequence described in preceding two kinds of schemes, perhaps cyclisation, perhaps L-type amino acid becomes D-type amino acid, perhaps disappearance, or add and the function coordinator polypeptide that obtains.
More excellent scheme is: front end, middle part or end contain any described antibacterial peptide aminoacid sequence of several schemes in front in the described antibacterial peptide aminoacid sequence.
Two of purpose of the present invention is also to disclose a kind of synthesis technique of artificial antibacterial peptide, adopts following technical scheme:
A kind of method of artificial synthetic antimicrobial peptide comprises the steps:
1) the mytilin B with the Mediterranean Sea mussel is a template, and Mytilus crassitesta Lischke mytilin-1 is carried out the space structure simulation; According to the space structure of mytilin-1, select No. 20 L-Ala (Ala20) to the decapeptide fragment between No. 29 halfcystines (Cys29), and Ala20 and Ser22 are replaced to Cys respectively, obtain a new polypeptide, called after MDP-1; And reverse sequence, called after MDP-2;
2) adopt solid-phase synthesis, utilize Fmoc-Cys (trt)-OH and Fmoc-Cys (Acm)-OH to synthesize required linear polypeptide as a pair of Cys that will form disulfide linkage respectively;
3) raw product that obtains being carried out renaturation with one or several mixing solutionss among iodine/methyl alcohol/DMF and xitix respectively handles to form correct disulfide linkage;
4) to RPLC (RP-HPLC) purifying at least 2 times of the polypeptide sample after the renaturation, obtain pure product.
Preferably: the N of MDP-1 or MDP-2 polypeptide end is designed to acetylize in the described step 1), and the C end is designed to amidation to remove terminal electric charge.
More preferably: solid-phase synthesis technology is described step 2):
21) the Fmoc-Rink-AM-Resin resin carries out abundant swelling with methylene dichloride (DCM); Getting first 9-fluorenylmethyloxycarbonyl (Fmoc)-amino acid dissolves in DCM; Add diisopropylethylamine (DIEA) again and mix in the back adding reaction vessel, blow N
2Reaction 2hr;
22) reacting liquid filtering is removed and added methyl alcohol, use DCM, Virahol and N behind the capping 1hr respectively, dinethylformamide (DMF) washing resin; Take off Fmoc with 20% piperidines afterwards, behind the DMF thorough washing, carry out coupling after adding second amino acid and 1-carbonyl benzo tetrazolium (HOBt) and DIEA mixing, react after one hour, detect with triketohydrindene hydrate, the resin water white transparency is for reacting completely, then with the DMF thorough washing;
23) repeat above-mentioned steps to the last an amino acid react completely;
24) utilizing K reagent (K-Reagent contains 82.5% trifluoroacetic acid, 5% phenol, 5% thioanisole, and the aqueous solution of 2.5% mercaptoethanol) that the polypeptide after synthetic is carried out side chain goes to protect and cut from resin.
Three of purpose of the present invention is to disclose with artificial synthetic antimicrobial peptide in preparation treatment gram-positive microorganism, Gram-negative bacteria, the drug development of fungi infestation or the application in the fodder additives.
Cys represents that English name is Cysteine, and Chinese is the corresponding residue of the amino acid of halfcystine, abbreviates C as.
Val represents that English name is Valine, and Chinese is the corresponding residue of the amino acid of Xie Ansuan, abbreviates V as.
Phe represents that English name is Phenylalanine, and Chinese is the corresponding residue of the amino acid of halfcystine, abbreviates F as.
Gly represents that English name is Glycine, and Chinese is the corresponding residue of the amino acid of glycine, abbreviates G as.
Arg represents that English name is Arginine, and Chinese is the corresponding residue of arginic amino acid, abbreviates R as.
Ile represents that English name is Isoleucine, and Chinese is the corresponding residue of the amino acid of Isoleucine, abbreviates I as.
The present invention compared with prior art, have following advantage and effect: the present invention adopts the method for synthetic, synthetic antibacterial peptide compare with natural antibacterial peptide have that molecular weight is little, Stability Analysis of Structures, anti-microbial activity is strong, sequence is short, few, the low cost and other advantages of disulfide linkage number.
Description of drawings
Accompanying drawing 1:M is according to the MDP-1 of the active region design of Mytilus crassitesta Lischke antibacterial peptide mytilin-1 and the sequence chart of MDP-2.
High performance liquid phase behind accompanying drawing 2:MDP-1 and the MDP-2 solid phase synthesis detects collection of illustrative plates.
Mass spectrometric detection collection of illustrative plates behind accompanying drawing 3:MDP-1 and the MDP-2 solid phase synthesis.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is described in further details:
Embodiment one: the solid state chemistry of antibacterial peptide is synthetic, separation and purification and evaluation
1. the artificial design of novel antibacterial peptide
Utilize structural simulation software ESyPred3D (Lambert C, Leonard N, De Bolle X, Depiereux E.ESyPred3D:Prediction of proteins 3D structures.Bioinformatics.2002 Sep; 18 (9): 1250-1256.), with mytilin B (the protein structure database numbering: 2EEM) be template, Mytilus crassitesta Lischke mytilin-1 is carried out the space structure simulation of Mediterranean Sea mussel (Mytilus galloprovincialis).According to the structural simulation result, we find, Mytilus crassitesta Lischke mytilin-1 has by two sections beta-pleated sheets equally and connects the ring of these two sections beta-pleated sheets, and be distributed with alkaline amino acid residue on the ring, space structure according to mytilin-1, select No. 20 L-Ala (Ala20) to the decapeptide fragment between No. 29 halfcystines (Cys29), and Ala20 and Ser22 replaced to Cys respectively, obtain a new polypeptide, called after MDP-1, the N end of this polypeptide is designed to acetylize, and the C end is designed to amidation to remove terminal electric charge, and its sequence is Ac-Cys-Val-Cys-Phe-Gly-Arg-Arg-Cys-Ile-Cys-NH2 as shown in Figure 1; Simultaneously, we have designed a reverse sequence in addition, called after MDP-2, and its sequence is Ac-Cys-Ile-Cys-Arg-Arg-Gly-Phe-Cys-Val-Cys-NH2.
2. the solid phase synthesis of novel antibacterial peptide
The present invention uses solid-phase synthesis (solid-phase peptide synthesis, SPPS), utilize the different properties of Fmoc-Cys (trt)-OH (CysI and Cys III) and Fmoc-Cys (Acm)-OH (Cys II and Cys IV), synthesize required linear polypeptide as a pair of Cys that will form disulfide linkage respectively, so that the correct pairing of disulfide linkage.
Solid-phase polypeptide synthesizes on Tetras Peptide synthesizer (U.S. ThuraMed company) and carries out, and basic procedure is: the Fmoc-Rink-AM-Resin resin carries out abundant swelling with methylene dichloride (DCM); Get first 9-fluorenylmethyloxycarbonyl (Fmoc)-amino acid and in DCM, dissolve, add diisopropylethylamine (DIEA) again and mix in the back adding reaction vessel, blow N
2Reaction 2hr removes adding methyl alcohol with reacting liquid filtering, uses DCM, Virahol and N behind the capping 1hr respectively, dinethylformamide (DMF) washing resin; Take off Fmoc with 20% piperidines afterwards, behind the DMF thorough washing, carry out coupling after adding second amino acid and 1-carbonyl benzo tetrazolium (HOBt) and DIEA mixing, react after one hour, detect with triketohydrindene hydrate, the resin water white transparency is for reacting completely, then with the DMF thorough washing; A repetition above-mentioned steps to the last amino acid reacts completely.Utilizing K reagent (K-Reagent contains 82.5% trifluoroacetic acid, 5% phenol, 5% thioanisole, and the aqueous solution of 2.5% mercaptoethanol) that the polypeptide after synthetic is carried out side chain goes to protect and cut from resin.
3. the renaturation of novel antibacterial peptide, purifying and evaluation
The synthetic crude product that obtains carries out renaturation with iodine/methyl alcohol/DMF mixing solutions and xitix respectively to be handled to form correct disulfide linkage (Cys I-Cys III, Cys II-Cys IV).
RPLC (RP-HPLC) purifying 2 times of polypeptide sample after the renaturation.Purifying for the first time: Waters Sunfire C18 post (10mm * 250mm), gradient: 0～40min, 5%～30% acetonitrile (containing 0.1%TFA); Purifying for the second time: Vydac C18 post (4.6mm * 250mm), gradient: 0～40min, 5%～20% acetonitrile (containing 0.1%TFA).The high performance liquid phase of MDP detects collection of illustrative plates and sees accompanying drawing 2, and the purity of MDP-1 and MDP-2 is all greater than 95% behind two-step purifying.
With the accurate molecular weight at Waters ZQ2000 spectrometer analysis purpose peak, detected result is seen accompanying drawing 3.The mass spectrometric detection condition is capillary voltage: 3.50KV; Taper hole voltage: 35V; Dry gas flow velocity: 500L/h; Ion source temperature: 120 ℃; Auxiliary temperature degree: 380 ℃; Ion detection mode: selectivity ion detection (SIM); Ion polarity: positive ion (positive); Ionization mode: pneumatic auxiliary electro-spray ionization (ESI).Through mass spectroscopy, single isotopic molecule amount ([M+H]+) of MDP-1 and MDP-2 is respectively 1197.34Da and 1197.46Da.Consistent with the molecular weight that theory is derived, show the success of synthetic and renaturation.
Embodiment two: the Function detection of antibacterial peptide
1. the antibacterial experiment of novel antibacterial peptide and antimicrobial spectrum
Adopt the substratum coubling dilution to measure MDP-1 and MDP-2 anti-microbial activity.Bacterium is cultured to logarithmic phase (A with the LB liquid nutrient medium
630nmBe 0.001), add 90 μ L bacterium liquid in each hole of 96 orifice plates.With PBS dissolving, maximum concentration is 1mM to polypeptide in advance, doubling dilution then, and minimum concentration is 6.25 μ M.And each concentration polypeptide solution added in 96 orifice plates, 10 μ L/ holes, PBS is as negative control.
The soft vibration on vibrator of 96 orifice plates is made sample and bacterium liquid thorough mixing, put into 37 ℃ then and cultivate 16-24h.Adopt microplate reader to measure the OD value in each hole to weigh the restraining effect of polypeptide for bacterial growth.The minimum inhibitory concentration of polypeptide (Minimal inhibitory concentrations, MIC) with [a]-[b] expression, wherein [a] represents the peak concentration of bacterium continued growth, and the minimum concentration that on behalf of bacterium, [b] be killed fully.Antimycotic test and above-mentioned basic identical, just fungi detects behind 30 ℃ of cultivation 48h in the Sabourand liquid nutrient medium.The result is as shown in table 1.
Table 1MDP-1 and MDP-2 are for the minimum inhibitory concentration (MIC) of various bacteriums and fungi
2.MDP stability test
Dissolve MDP-1 and MDP-2 respectively with pure water, concentration is 100 micromoles, place 37 ℃ of thermostat containers to preserve, sampling in per 24 hours once, carry out mass spectrum evaluation and anti-microbial activity test respectively, and compare with the dry powder MDP-1 and the MDP-2 of-20 ℃ of preservations, with the stability of the 26S Proteasome Structure and Function of check novel antibacterial peptide.Experimental result shows, at 37 ℃, under the pure water preservation condition, MDP-1 and MDP-2 still have stronger bacteriostatic action after preserving 20 days, (the dry powder MDP-1 of 20 ℃ of preservations and MDP-2) compares with control group, and its antibacterial effect is declined by less than 5%, and mass spectrometric detection still is single mass peak, this shows that MDP-1 and MDP-2 all have structure and functional stabilization preferably.
3. external hemolytic activity detects
Present embodiment is used to detect synthetic antibacterial peptide whether human erythrocyte is had hemolytic activity.The detection step is as follows:
Gather in about 0.5mL to the 5mL PBS of the normal human blood solution, owing to blood is fully diluted, thereby even without using the heparin processing still aggegation can not take place.At 4 ℃, the centrifugal 5min of 3000rpm repeats Washed Red Blood Cells fully 3 times.Estimate sedimentary volume, add the resuspended red corpuscle of PBS, the red corpuscle final concentration is about 1%.The red corpuscle PBS solution of dilution is added 1.5mL EP pipe, 50 μ L/ pipe; Each EP pipe adds the antibacterial peptide PBS solution of doubling dilution in advance then, 50 μ L/ pipe, then cumulative volume is 100 μ L, the final concentration of antibacterial peptide sample is 1000 μ M, negative control adds 50 μ L PBS solution, and the positive control of complete hemolysis adds the TritonX-100 of 50 μ L 0.1%.Hatch the centrifugal 5min of 12000rpm behind the 1hr for 37 ℃, supernatant carefully is taken to 96 orifice plates, utilize microplate reader to measure the OD value of supernatant when 570nm.Calculate erythrocytic hemolytic activity by following formula: haemolysis %=(A
Sample-A
Negative)/(A
Positive-A
Negative).
Experimental result shows, under 1000 μ M concentration, MDP-1 and MDP-2 can induce about 10% red corpuscle haemolysis to occur, illustrate that MDP is lower to erythrocytic haemolysis effect, and this shows that also MDP is lower to the toxicity of mammalian cell.
A kind of artificial synthetic polypeptide and sequence table thereof
＜110〉Oceanography Institute Of Zhejiang's ocean science institute
＜120〉the artificial design of two kinds of antibacterial peptides, the synthetic and application of solid state chemistry
<160>3
<170>PatentIn?version?3.5
<210>1
<211>10
<212>MDP-1?peptide
＜213〉artificial sequence
<400>1
Cys?Val?Cys?Phe?Gly?Arg?Arg?Cys?Ile?Cys
＜110〉Oceanography Institute Of Zhejiang's ocean science institute
＜120〉the artificial design of two kinds of antibacterial peptides, the synthetic and application of solid state chemistry
<160>3
<170>PatentIn?version?3.5
<210>2
<211>10
<212>MDP-2peptide
＜213〉artificial sequence
<400>2
Cys?Ile?Cys?Arg?Arg?Gly?Phe?Cys?Val?Cys
Claims (8)
1. artificial synthetic antimicrobial peptide, it is characterized in that: described antibacterial peptide aminoacid sequence is Cys-Val-Cys-Phe-Gly-Arg-Arg-Cys-Ile-Cys; Perhaps be Cys-Ile-Cys-Arg-Arg-Gly-Phe-Cys-Val-Cys.
2. artificial synthetic antimicrobial peptide as claimed in claim 1 is characterized in that: comprise two pairs of disulfide linkage in the described antibacterial peptide aminoacid sequence, described antibacterial peptide has β-hairpin structure.
3. artificial synthetic antimicrobial peptide, it is characterized in that: described antibacterial peptide is one or several amino acid whose being substituted in claim 1 or the 2 described antibacterial peptide aminoacid sequences, perhaps cyclisation, perhaps L-type amino acid becomes D-type amino acid, perhaps disappearance, or add and the function coordinator polypeptide that obtains.
4. artificial synthetic antimicrobial peptide, it is characterized in that: front end, middle part or end contain each described antibacterial peptide aminoacid sequence of claim 1-3 in the described antibacterial peptide aminoacid sequence.
5. the method for an artificial synthetic antimicrobial peptide comprises the steps:
1) the mytilin B with the Mediterranean Sea mussel is a template, and Mytilus crassitesta Lischke mytilin-1 is carried out the space structure simulation; According to the space structure of mytilin-1, select No. 20 L-Ala (Ala20) to the decapeptide fragment between No. 29 halfcystines (Cys29), and Ala20 and Ser22 are replaced to Cys respectively, obtain a new polypeptide, called after MDP-1; And reverse sequence, called after MDP-2;
2) adopt solid-phase synthesis, utilize Fmoc-Cys (trt)-OH and Fmoc-Cys (Acm)-OH to synthesize required linear polypeptide as a pair of Cys that will form disulfide linkage respectively;
3) raw product that obtains being carried out renaturation with one or several mixing solutionss among iodine/methyl alcohol/DMF and xitix respectively handles to form correct disulfide linkage;
4) to RPLC (RP-HPLC) purifying at least 2 times of the polypeptide sample after the renaturation, obtain pure product.
6. the method for artificial synthetic antimicrobial peptide as claimed in claim 5 is characterized in that: the N of MDP-1 or MDP-2 polypeptide end is designed to acetylize in the described step 1), and the C end is designed to amidation.
7. as the method for claim 5 or 6 described artificial synthetic antimicrobial peptides, it is characterized in that: solid-phase synthesis technology is described step 2):
21) the Fmoc-Rink-AM-Resin resin carries out abundant swelling with methylene dichloride (DCM); Getting first 9-fluorenylmethyloxycarbonyl (Fmoc)-amino acid dissolves in DCM; Add diisopropylethylamine (DIEA) again and mix in the back adding reaction vessel, blow N2 reaction 2hr;
22) reacting liquid filtering is removed and added methyl alcohol, use DCM, Virahol and N behind the capping 1hr respectively, dinethylformamide (DMF) washing resin; Take off Fmoc with 20% piperidines afterwards, behind the DMF thorough washing, carry out coupling after adding second amino acid and 1-carbonyl benzo tetrazolium (HOBt) and DIEA mixing, react after one hour, detect with triketohydrindene hydrate, the resin water white transparency is for reacting completely, then with the DMF thorough washing;
23) repeat above-mentioned steps to the last an amino acid react completely;
24) utilizing K reagent that the polypeptide after synthetic is carried out side chain goes to protect and cut from resin.
8. each described artificial synthetic antimicrobial peptide of claim 1-4 is in preparation treatment gram-positive microorganism, Gram-negative bacteria, the drug development of fungi infestation or the application in the fodder additives.
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